This proposal is directed to the study of the interactions of the histone structural unit, the nucleosome, with DNA during chromatin replication. Nascent DNA-nucleosome interactions will be studied by several different methodologies, both in vivo and in vitro. The mechanism of distribution of parental nucleosomes to daughter chromatids will be investigated by analysis of pulse-labeled, nascent chromatin DNA by micrococcal nuclease cleavage. Preliminary data is presented for a comprehensive investigation to determine this mechanism. The time of replicon maturation, i.e., whether nucleosomes are added to the replication eye prior to, or subsequent to termination of DNA synthesis will be studied. A technique for the isolation of replication eye chromatin is described. An in vitro DNA synthesis system with intact nuclei will be employed for investigation of whether nucleosomes are freed from DNA during DNA synthesis, or whether DNA synthesis occurs on nucleosome-associated DNA, without protein displacement. We will determine whether Okazaki fragments are associated with nucleosomes, whether the termini of these DNA precursor fragments are internal or external to nucleosome-associated nascent DNA, and whether gap-filling and ligation of nascent DNA occur on nucleosome-associated nascent DNA.